Multidrug resistance (MDR) mediated by P-glycoprotein remains one of the most difficult problems in cancer research and in the care of patients with drug-resistant cancer. During the last grant period we undertook a series of experiments designed to gain a more detailed understanding of the factors that regulate the expression of the MDR1 gene and to attempt to understand the behavior of MDR cancers. The results of this work opened several exciting areas for future investigation. First, we found that signaling from the cell surface to the nuclear transcription of MDR1 was mediated by phospholipase C and that the MDR1 gene expression could be activated by factors that increased signaling through this pathway and inhibited by agents that blocked one of several signal transduction components. Next, we pursued our original observations on the metastatic behavior of MDR cell lines and found that drugs transported by P-gp could increase cell motility and invasion through a pathway mediated by phosphatidylinositol-3 kinase. In addition, we found that MDR cancer cell lines produced greater quantities of metalloproteinases than parental cell lines and that these activities appeared to be regulated by EMMPRIN (extra cellular matrix metalloproteinase inducers), a cell surface glycoprotein that regulates the activities of matrix metalloproteinases from stromal and tumor cells. When studying the features of MDR cells that predispose to metastases, we carried out 2-D gel characterization and found that MDR cancer cells overexpressed ubiquitin carboxyl-terminal hydrolase L1, an enzyme that plays a critical role in protein stability by replenishing intracellular ubiquitin stores. This led us to evaluate the role of ubiquitination in MDR and found that glycosylation regulated the ubiquitination State of P-gp and that P-gp appears to undergo ubiquitin-mediated degradation. During the next grant period we plan to pursue each of these exciting and relatively unexplored areas of MDR research to ultimately gain a better understanding of how to manage patients with drug resistant cancer.